Expression and characterization of sucrose synthase from mung bean [Vigna radiata] seedlings in Escherichia coli

• Published in: Bioscience, biotechnology, and biochemistry Vol. 61; no. 9; pp. 1500 - 1503
• Main Authors: Nakai, T. (Kyoto Univ., Uji (Japan). Wood Research Inst.), Tonouchi, N, Tsuchida, T, Mori, H, Sakai, F, Hayashi, T
• Format: Journal Article
• Language: English
• Published: Tokyo Taylor & Francis 01.09.1997; Japan Society for Bioscience Biotechnology and Agrochemistry; Oxford University Press
• Subjects: ADN RECOMBINADO; ADN RECOMBINE; Analytical, structural and metabolic biochemistry; Biological and medical sciences; Biotechnology; Blotting, Western; Chromatography, Gel; Cloning, Molecular; Electrophoresis, Polyacrylamide Gel; Enzymes; Enzymes and enzyme inhibitors; ESCHERICHIA COLI; Escherichia coli - enzymology; Fabaceae - enzymology; Fundamental and applied biological sciences. Psychology; Glucosyltransferases - analysis; Glucosyltransferases - biosynthesis; Kinetics; LIGASAS; LIGASE; LIGASES; Metabolism; mung bean; Plant physiology and development; Plants, Medicinal; PLANTULAS; PLANTULE; Plasmids - genetics; recombinant; RECOMBINANT DNA; Recombinant Proteins - biosynthesis; SACCHAROSE; SEEDLINGS; SUCROSA; SUCROSE; sucrose synthase; Transferases; VIGNA RADIATA; Heterologous system; Sucrose; Enzyme; Transferases; Escherichia coli; Glycosyltransferases; Vigna radiata; Biosynthesis; Gene expression; Native state; Grain legume; Leguminosae; Enzymatic activity; Dicotyledones; Tetramer; Angiospermae; Bacteria; Hexosyltransferases; Spermatophyta; Kinetic parameter; Recombinant protein; Sucrose synthase; Comparative study; Enterobacteriaceae
• ISSN: 0916-8451; 1347-6947
• DOI: 10.1271/bbb.61.1500

The cDNA fragment coding for mung bean (vigna radiata Wilczek) sucrose synthase was introduced into the expression vector pET-20b resulting in the construction of plasmid pEB-01. After transformation of Escherichia coli strain BL21(DE3) cells by pEB-01 and induction with isopropyl thio-beta-galactoside, high level expression of the recombinant enzyme was obtained. The enzyme had a tetrameric form that conserved the activity of sucrose synthase. Although the Km and Vmax of the recombinant enzyme acting on either UDP-glucose or fructose were very close to those of the native enzyme isolated from mung bean seedlings, the Km for sucrose was higher by a factor of 10 for the recombinant enzyme. This suggests that the recombinant sucrose synthase has a tendency to synthesize sucrose, although the native enzyme catalyzes a freely reversible reaction